Serum matrix metalloproteinase‐7, Syndecan‐1, and CA 19‐9 as a biomarker panel for diagnosis of pancreatic ductal adenocarcinoma

Abstract Aims and Background Matrix metalloproteinase‐7 (MMP‐7) and Syndecan‐1 (SDC1) are involved in multiple functions during tumorigenesis. We aimed to evaluate the diagnostic and prognostic performance of these serum proteins, as potential biomarkers, in patients with pancreatic ductal adenocarcinoma (PDAC) and benign pancreatic cysts. Methods In this case–control study, patients with newly diagnosed PDAC (N = 121) were compared with the benign cyst (N = 66) and healthy control (N = 48) groups. Serum MMP‐7 and SDC1 were measured by ELISA. The diagnostic accuracy of their levels for diagnosing PDAC and pancreatic cysts was computed, and their association with survival outcomes was evaluated. Results MMP‐7 median serum levels were significantly elevated in the PDAC (7.3 ng/mL) and cyst groups (3.7 ng/mL) compared with controls (2.9 ng/mL) (p < 0.001 and 0.02, respectively), and also between the PDAC and cyst groups (p < 0.001), while SDC1 median serum levels were significantly elevated in PDAC (43.3 ng/mL) compared with either cysts (30.1 ng/mL, p < 0.001) or controls (31.2 ng/mL, p < 0.001). The receiver operating characteristic curve analysis area under the curve in PDAC versus controls was 0.90 and 0.78 for MMP‐7 and SDC1, respectively, while it was 1.0 for the combination of the two and CA 19‐9 (p < 0.001). The combination of the three biomarkers had a perfect sensitivity (100%). Conclusions Due to its high sensitivity, this biomarker panel has the potential to rule out PDAC in suspected cases.


| INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death, with an estimated five-year survival rate of only 13% despite the improvements in therapeutic strategies. 1 Its poor prognosis relates not only to an intrinsic biological aggressiveness, but also to late presentation of clinical symptoms as well as lack of reliable strategies for early detection in average-risk population. 2 While most PDAC develop from Pancreatic Intraepithelial Neoplasia (PanIN), about 15%-20% grows from welldefined pre-malignant cystic lesions, namely intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs). 2,3In the absence of biomarkers with sufficient diagnostic accuracy, current pancreas neoplasia screening relies on imaging or endoscopic ultrasonography (EUS) to differentiate between low-risk and high-risk lesions for developing PDAC. 4 Matrix metalloproteinases (MMPs) comprise a family of at least 25 secreted and cell surface zinc-dependent endopeptidases that are involved in physiological and pathological remodeling of extracellular matrix, 5 but also in regulating signaling pathways that control cell growth, and as such are also involved in tumor progression and invasion of surrounding tissue. 6,7Specifically, MMP-7 is overexpressed in PanIN and was shown to be upregulated approximately 10-fold in pancreatic cancer. 8][13] Syndecan-1 (SDC1) is a member of the transmembrane heparan sulfate proteoglycan family and is one the proteins that are released by MMP-7 to the serum.Although primarily involved in cell adhesion, migration, and cellmatrix interactions, 14,15 it also regulates multiple functions during tumorigenesis, including tumor cell attachment, proliferation, and angiogenesis through different signaling pathways (e.g., Wnt pathway activation). 16,17In the era of PDAC, two recent published studies have linked SDC1 levels with mutated overexpressed KRAS, the initiating step in most PDACs, which cooperate to induce a malignant phenotype through regulation of macropinocytosis, a critical metabolic pathway that fuels PDAC cell growth and promotes tumor progression. 18,19Moreover, we have recently shown elevated baseline serum SDC1 levels in PDAC compared to healthy individuals, 20 potentially serving as a diagnostic biomarker.
Although these two proteins were previously evaluated as potential biomarkers in PDAC, they have not been hitherto evaluated in combination in PDAC or in pre-malignant cystic lesions, neither was their combination with CA 19-9 explored for a potential increased diagnostic accuracy.We thus aimed to evaluate the diagnostic and prognostic performance of serum MMP-7 and SDC1 as potential biomarkers, separately and combined, and as adjunct to CA 19-9, in patients with PDAC and pancreatic cysts.

| Design and patient population
This was a case-control study conducted at the Sheba Medical Center, a tertiary academic center in Israel.We included two prospectively recruited study populations: (1) Patients with newly diagnosed PDAC by either EUSguided biopsy or by surgically obtained specimen.(2)  Patients with newly diagnosed pancreatic cysts, based on either EUS evaluation or surgical resection, coupled with subsequent follow-up history.The serum samples from all patients were obtained from two sources: (1) The Sheba Medical Center's Tissue Bank Repository for PDAC patients and those with pancreatic cysts who underwent upfront surgery between September 2014 and December 2022; (2) The Gastrointestinal (GI) Institute for patients with newly diagnosed EUS-based disease between October 2019 and December 2022.Healthy controls were recruited after visiting the GI clinics for general symptoms.All patients were followed up through December 2022.
In all patients, baseline serum markers samples obtained before any surgical or oncological intervention were tested for the tumor marker CA 19-9, SDC1, and MMP-7.The data collected from medical files included demographic characteristics, presence of diabetes, smoking habits, tumor/cyst location, germline testing (if performed), cyst characteristics, pathological/clinical staging, surgery conduction, and date of censor (December 2022)/ death.Exclusion criteria from the study included patients who were unable to sign an informed consent (see below), patients who suffered from autoimmune disorders, systemic infections, or extra-pancreatic malignancies.All patients provided an informed consent (either for this study or for the central tissue banking), Sheba institutional ethics review board approved the study (SMC-6185-19).

| Definitions and classifications
PDAC staging was classified either by three clinically distinct patient groups: resectable (T1-3, stages 1 and 2), locally advanced (T4, stage 3), and metastatic disease (M1, stage 4), or by the American Joint Committee on Cancer (AJCC)/TMN system staging for PDAC.Determination of staging was based on either pathological assessment for resectable ones or imaging modalities (mainly CT scans) for non-resectable tumors.We considered preoperative staging for the purpose of categorization for locally advanced/borderline tumors in which neo-adjuvant treatment was given before surgery.
For cystic lesions, worrisome features and high-risk stigmata for IPMN were defined in this study according to the Sendai and Fukuoka International Consensus Guidelines for management of mucinous cysts. 4For surgically removed lesions, high-grade precursor lesions were defined as PanIN-3 /IPMN with high-grade dysplasia.All other lesions (namely, benign cystic lesions with no highrisk stigmata, or low-grade precursor lesions like PanIN-1, PanIN-2, and IPMN with low-or moderate-grade dysplasia) were considered non-significant.

| Soluble Syndecan-1 analysis
Serum samples were collected, centrifuged at 3000 rpm for 10 min and stored at −80°C.Serum SDC1 levels were evaluated using human SDC1 ELISA (Diaclone Research, Besancon, France) according to the instructions provided by the manufacturer.The standards and samples were analyzed in duplicate.Concentrations of Serum SDC1 were reported as ng/mL.The technicians were blinded to the clinical data.

| Serum matrix metalloproteinase-7analysis
Serum samples were collected, centrifuged at 3000 rpm for 10 min and stored at −80°C.Concentrations of serum MMP-7 were evaluated using human MMP-7 ELISA (R&D systems, Minneapolis, MN, USA) following the manufacturer's instructions, using standards provided with the kit.Standards and samples were analyzed in duplicate.Serum MMP-7 levels were reported as ng/mL.The technicians were blinded to the clinical data.

| CA 19-9 analysis
Serum CA 19-9 was quantitatively determined by the Access® GI Monitor assay on the Beckman Coulter Unicell DXI 800 following the clinical and laboratory standards institute guidelines.

| Statistical analysis
Categorical variables were summarized as frequencies and percentages.The distribution of continuous variables was evaluated using histograms.Since all continuous variables were not normally distributed, they were reported as median and interquartile range (IQR).The Mann-Whitney Utest was used to compare the continuous variables between groups and chi-squared test was used to compare the categorical variables between the two groups.The area under the Receiver operating characteristic (ROC) curve was used to evaluate the ability of the biomarkers to discriminate between the patient groups.The areas under the ROC curve were compared using DeLong test.Logistic regression was used to evaluate the patients' probabilities of having cancer based on biomarkers.Chi-squared automatic interaction detection (KASS 1980) was used to identify subgroups of patients.Spearman's correlation coefficient was used to study the correlation between the biomarkers.Log-rank test and Kaplan-Meier curve were used to compare the association between the biomarkers categories and patients' survival.All statistical tests were 2-sided, and p < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS version 28 (IBM, Armonk, NY, USA).

| Patient characteristics
The characteristics of the study population are depicted in Table 1.The median (IQR) age was 70 (64-75), 70 (65-75), and 67 (59-73) for the PDAC (N = 121), cyst (N = 66), and control (N = 48) groups (p = 0.08), respectively.The rates of diabetes and smoking were significantly higher in the PDAC group than in the cyst and control groups, and male gender was more frequent among the PDAC group (p = 0.06).
Using ROC analysis for PDAC, a cutoff level of 3.5 ng/ mL serum MMP-7 showed a sensitivity of 92% and a specificity of 65%, while cutoff level of 30 ng/mL serum

| DISCUSSION
In this study, we found that a combination of three biomarkers, namely serum MMP-7, SDC1, and CA 19-9, provided superior diagnostic accuracy for PDAC compared with any of these biomarkers alone, with a perfect sensitivity to PDAC.Moreover, MMP-7 levels correlated with more advanced, metastatic disease and the combination of MMP-7 and SDC1 demonstrated prognostic value in PDAC.MMP-7 was previously assessed in several studies and found to be associated with metastatic PDAC.][10][11] Serum levels were also assessed for their utility for predicting severity of disease and survival.In one surgical study, MMP-7 had modest utility to predict unresectability and nodal involvement (AUC = 0.68), while for patients with levels above 13.5 ng/mL (approximately 15% of the cohort), it was highly predictive for unresectable disease. 12In another study, all PDAC patients without lymph node involvement had MMP-7 serum levels <20 ng/mL, versus levels >20 ng/mL in all metastatic patients. 11Moreover, the combined monitoring of serum MMP-7 and CA 19-9 improved the predictive value for PDAC compared with MMP-7 alone, yielding AUC of 0.90-0.99. 13,21In comparison to these studies, our results show AUC of 0.90 and 0.99 for MMP-7 exclusively and in combination with CA 19-9, respectively.In addition to its diagnostic utility, MMP-7, separately and combined with SDC1, had prognostic value in PDAC.Notably, the cutoff of MMP-7 for its diagnostic and prognostic values differed, being 3.5 and 10 ng/mL, respectively.
In contrary to MMP-7, there is limited data of the role of SDC1 in PDAC.SDC1 was first shown to be overexpressed in pancreatic tissues from patients with progression to PDAC. 22,23However, two recent studies have linked its pathogenic role to mutated KRAS, 18,19 the initiating step in the majority of PDACs, which contributes to the induction of malignant phenotype.In a landmark study by Yao and colleagues, in transgenic mice models of PDAC, oncogenic KRAS induced SDC1 cell surface overexpression, where it regulated macropinocytosis, a crucial metabolic pathway that fuels PDAC cell growth and promotes tumor progression. 18Recently, Zhang et al. found that the expression of SDC1 indicated a poor survival in PDAC patients and confirmed that depletion of SDC1 can significantly suppress in-vitro PDAC cell proliferation, induce cell apoptosis, and impair cell migration. 24hus, the combined use of MMP-7 and SDC1 has pathophysiologic rationale.MMP-7 was demonstrated to shed SDC1 complexes with CXC chemokine from cell surfaces, where it was established in spatially localizing neutrophil activation to epithelia. 25,26Since the MMP-7induced shedding of SDC1 ectodomain was previously observed in cultured pancreatic cancer cell lines, 27 it is possible that the increased serum SDC1 level in our cohort was related to MMP-7.Indeed, in our study we observed a correlation between serum levels of these two proteins, as expected.However, the superior sensitivity and yield of serum MMP-7 levels over SDC1 in PDAC, suggests that it may be the dominant modulator in tumor progression and microenvironment, possibly thorough several mechanisms of which one is the release of SDC1 ectodomain.Our observation that the combined use of MMP-7 and SDC1 modestly increased diagnostic sensitivity for PDAC and that some patients had only elevated serum SDC1 level, may suggest additional mechanisms for its shedding other than cleavage by MMP.These may include tumor-induced overexpression of cell surface SDC1 28 or upregulation of heparanase, an endoglycosidase that specifically degrades the heparan-sulfate chains of SDC1. 29,30The potential effect of MMP-7-induced shedding of SDC1 on macropinocytosis in PDAC remains to be clarified.
The combined three-biomarkers panel showed the potential optimal diagnostic yield for PDAC.Although shown in a small cohort, a sensitivity of 100% could make it an ideal panel for ruling out PDAC in suspected cases.However, in light of the fact that most of our cohort (>80%) had advanced or metastatic disease, the utility for early detection of the panel could not be assessed.Nonetheless, additional diagnostic tools, including imaging and bloodbased biomarkers, are being developed and we hypothesize that a combination panel (blood based and imaging) will perform the best strategy for early detection.Notably, despite an isolated specificity of 100% for CA 19-9 in our study, probably affected by the relatively small study sample of healthy individuals, previous studies showed a more limited diagnostic accuracy as well, with PPV between 80% and 90%. 31ur study is the first to show that benign cystic pancreatic lesions were associated with significantly higher serum levels of MMP-7 than controls.In contrast, serum levels were not different between low-versus high-risk cystic lesions, although this observation must be interpreted cautiously, considering the small patient number with high-risk features.Importantly, other benign pre-malignant pancreatic lesions, for example, chronic pancreatitis (CP) and benign peri-pancreatic neoplasms (duodenal/papillary adenomas), were not associated with high serum levels of MMP-7. 13However, upregulated tissue expression was demonstrated in patients with these pre-malignant pancreatic lesions (CP and metaplastic duct lesions). 8Although cystic MMP-7 was not measured in our study, its high serum level thus supports the existence of upregulated tissue expression within pre-malignant cysts with spillage to the serum.SDC1, notably was one of the differentially expressed genes identified in IPMN lesions. 24The observation that it was not translated to significantly elevated serum levels in our study may be due to several possible factors, such as low level protein production by pre-cancerous cells, low level production of cells that constitutively express SDC1 that have been shed into the circulation or differential access to the circulation.Further studies should elucidate this point as well whether other precancerous lesions like mucinous cysts and PanIN, do express MMP-7 and SDC1.
The limitations to our study include serum MMP-7 and SDC1 were measured in a single time-point before any treatment, which limits our ability to study its prognostic role and its correlation to treatment.Also, we did not check for Lewis antigen status in our cohort, which may limit the diagnostic yield of CA 19-9.Another limitation was the lack of zymography in MMP-7 analysis of the serum samples.Using ELISA, these molecules are measured in totality, whereas, zymography may reveal, individually, the activated forms and degradation products of MMP-7.A small cohort of patients with high-grade cysts prevented us from showing a potential grading of serum level within this group.Finally, extrapolation to more heterogeneous populations need to be shown, since our cohort is from a relatively homogenous Israeli population.
In conclusion, this study shows the potential diagnostic use of a combination of serum biomarkers, consisting of MMP-7, SDC1, and CA 19-9, for the detection of PDAC, as all patients expressed one of these proteins in excess in their blood.Moreover, serum MMP-7 was modestly elevated in benign cystic lesions, although no difference was shown between low-and high-grade lesions, limiting its utility in this setting.However, more large-scale, and prospective studies should be performed to assess MMP-7 and SDC1, exact pathogenetic role in the development of these lesions and whether it can differentiate between simple and more high-risk lesions, potentially needing closer follow-up or surgery.Lastly, these combinatorial markers may be of value, as diagnostic biomarkers in carriers with high-risk potential to develop PDAC, for example, BRCA carriership or Peutz-Jehger.Future studies on high-risk groups in international consortium would be a preferential platform to explore this biomarker panel.

T A B L E 1
Abbreviation: PDAC, pancreatic ductal adenocarcinoma.
Box-plot representation of median serum matrix metalloproteinase-7 levels (ng/mL) in patients with pancreatic ductal adenocarcinoma (PDAC) according to disease progression.(B) Box-plot representation of median serum Syndecan-1 levels (ng/mL) in patients with PDAC according to disease progression.(C) Box-plot representation of median CA 19-9 levels (U/mL) in patients with PDAC versus cysts versus healthy controls.F I G U R E 3 (A) Receiver operating characteristic curve (ROC) of serum Syndecan-1, serum matrix metalloproteinase-7 (MMP-7), and CA 19-9, in single and combined, for diagnosis of pancreatic ductal adenocarcinoma (PDAC) versus healthy controls.(B) ROC curve of serum Syndecan-1, serum MMP-7, and CA 19-9, in single and combined, for diagnosis of PDAC versus pancreatic cyst.(C) ROC curve of serum Syndecan-1, serum MMP-7, and CA 19-9, single and combined, for diagnosis of pancreatic cysts versus healthy controls.T A B L E 2 Diagnostic accuracies of the serum biomarkers for PDAC versus control populations.

F
I G U R E 4 (A) Kaplan-Meier curve of overall 3-year survival among patients with serum matrix metalloproteinase-7 (MMP-7) levels >10 ng/mL at baseline versus those with baseline serum MMP-7 levels ≤10 ng/mL.(B) Kaplan-Meier curve of overall 3-year survival among patients with serum Syndecan-1 levels ≥35 ng/mL at baseline versus those with baseline serum Syndecan-1 levels <35 ng/m.(C) Kaplan-Meier curve of overall 1-year survival among patients with combined serum Syndecan-1 levels ≥35 ng/mL at baseline and baseline serum MMP-7 levels >10 ng/mL versus those with lower combined levels.